Cytokine signalling involves a cascade of intracellular intermediates activated by tyrosine phosphorylation and is tightly regulated. Signalling is initiated following cytokine binding to specific cell-surface receptors, inducing receptor oligomerisation. This allows transphosphorylation of receptor-associated Janus kinases (JAKs), which in turn phosphorylate the intracellular subunits of the receptor at specific tyrosine residues (Ihle et al., 1995). The phosphorylated tyrosines then act as docking sites for members of the signal transduction and activators of transcription (STAT) family. The STAT family is phosphorylated by the JAKs following docking, whereupon they dimerise and translocate into the nucleus, to function as transcription factors (Leonard and O'Shea, 1998).
The Suppressor of cytokine signalling proteins, SOCS1-7 and cytokine-inducible SH2-containing protein (CIS) (Starr et al., 1997; Yoshimura et al., 1995, Naka et al., 1997), not only directly disrupt the cytokine-induced intracellular signalling cascade, but also affect signal transduction by accelerating the turnover of signalling intermediates through the SOCS box (Hilton et al., 1998), which interacts with elonginB/C (Zhang et al., 1999) and cullin5 (Kamura et al., 2004) to form an E3 ubiquitin ligase. Transcription of SOCS1-3 and CIS, is upregulated following STAT activation (Hilton, 1999) and these SOCS proteins therefore control the duration of the signalling response via a negative feedback mechanism.
Socs3 knockout mice die in utero due to placental defects (Roberts et al., 2001). However, conditional knockout studies have illustrated that SOCS3 plays an indispensable role in regulating the inflammatory response and metabolism. For example, SOCS3 is essential in controlling the response to IL-6 (Croker et al., 2003; Lang et al., 2003; Yasukawa et al., 2003) and G-CSF (Croker et al., 2004; Kimura et al., 2004). Mice with haematopoietic deletion of Socs3 display a number of inflammatory disorders (Croker et al., 2004) and are acutely sensitive to G-CSF stimulation. Conditional knockout of Socs3 in neural cells leads to a severe loss in bodyweight via enhanced leptin signalling (Mori et al., 2004), whilst SOCS3 deficient adipocytes are protected against TNFα-induced insulin resistance (Shi et al., 2004). Intracellular delivery of SOCS3 reduces the production of inflammatory cytokines and attenuates liver apoptosis and haemorrhagic necrosis (Jo et al., 2005) in mice.
Similar to other members of the SOCS family, SOCS3 contains an N-terminal region, a central SH2 domain and a C-terminal SOCS box (see FIG. 1). The SH2 domain is responsible for direct or competitive inhibition of signalling proteins by interacting with the JAKs or blocking STAT access to docking sites on the receptors (Hilton, 1999; Kile et al., 2002). Although SOCS3 can interact directly with JAK2 via its SH2 domain (Sasaki et al., 2000), the highest-affinity binding sites for the SH2 domain are phosphorylated tyrosines on receptor subunits such as the IL-6 signalling subunit gp130, leptin and EPO receptors (Nicholson et al., 2000; Sasaki et al., 2000; Schmitz et al., 2000; Friederichs et al., 2001).
Extensive mutagenesis experiments involving SOCS3 (Sasaki et al., 1999; Yasukawa et al., 1999) have shown that regions outside the SH2 domain are required for high-affinity binding to phosphorylated tyrosines. These sequences, which extend 12 residues upstream and 40 residues downstream of the SH2 domain, were designated N- and C-ESS (extended SH2 subdomain) regions, respectively. Mutagenesis also identified a 12-residue region upstream of the N-ESS, the Kinase Inhibitory Region (KIR) that was required for kinase inhibition. Over-expression studies have shown that mutations in the SH2 domain and the KIR that abolished SOCS3 interaction with JAK also abrogated inhibition of STAT activity (Sasaki et al., 1999), but it is not clear how important a direct interaction between SOCS3 and JAK2 is in vivo. It is possible that SOCS3 only interacts directly with JAK when bound to sites on JAK-associated receptors such as gp130 (Yoshimura et al., 2005). Whether this then allows interaction with JAKs by the KIR and/or the SH2 domain remains unclear. To date there is no structural information available for any member of the SOCS family to allow an understanding of the molecular mechanism of JAK or STAT inhibition.
There is a need for a greater understanding of the structure of SOCS proteins, such as SOCS3. In particular, a greater appreciation of the structure/functional relationship is required to allow the design of molecules which can be used to modulate SOCS activity.